Effect of long-term methylene blue treatment on the composition of mouse gut microbiome and its relationship with the cognitive abilities of mice.

In recent years, methylene blue (MB) has attracted considerable interest as a potential drug for the treatment of methemoglobinemia and neurodegenerative diseases. MB is active against microorganisms from various taxonomic groups. However, no studies have yet been conducted on the effect of MB on the intestinal microbiome of model animals. The aim of this work was to study the effect of different concentrations of MB on the mouse gut microbiome and its relationship with the cognitive abilities of mice. We showed that a low MB concentration (15 mg/kg/day) did not cause significant changes in the microbiome composition. The Bacteroidetes/Firmicutes ratio decreased relative to the control on the 2nd and 3rd weeks. A slight decrease in the levels Actinobacteria was detected on the 3rd week of the experiment. Changes in the content of Delta, Gamma, and Epsilonproteobacteria have been also observed. We did not find significant alterations in the composition of intestinal microbiome, which could be an indication of the development of dysbiosis or other gut dysfunction. At the same time, a high concentration of MB (50 mg/kg/day) led to pronounced changes, primarily an increase in the levels of Delta, Gamma and Epsilonproteobacteria. Over 4 weeks of therapy, the treatment with high MB concentration has led to an increase in the median content of Proteobacteria to 7.49% vs. 1.61% in the control group. Finally, we found that MB at a concentration of 15 mg/kg/day improved the cognitive abilities of mice, while negative correlation between the content of Deferribacteres and cognitive parameters was revealed. Our data expand the understanding of the relationship between MB, cognitive abilities, and gut microbiome in respect to the antibacterial properties of MB.

Long-term mildronate treatment increased Proteobacteria level in gut microbiome, and caused behavioral deviations and transcriptome change in liver, heart and brain of healthy mice.

Mildronate is a cardiac and neuroprotective drug that is widely used in some countries. By inhibiting carnitine biosynthesis, mildronate impairs the fatty acids transport into mitochondria, thereby decreasing the ß-oxidation intensity. Since 2016, it has been prohibited by the World Anti-Doping Agency (WADA). However, the information on its safety and its influence on the athletes' health is scarce. There are no published studies on whether mildronate-induced long-term metabolism "rearrangement" may cause negative effects on high-metabolic-rate organs and on the whole organism. Here, we demonstrate that long-term mildronate treatment of healthy mice induced global metabolism change at the transcriptome level in liver, heart, and brain. Mildronate treatment also induced some behavioral changes such as anxiety-related behavior and diminished explorative behavior. We also found that mildronate induced a dysbiosis, as manifested by an increase in Proteobacteria level in gut microbiome. At the same time, the absence of a statistically significant increase in mouse strength and endurance procedures suggests that mildronate effect on productivity is negligible. The sum of our data suggests that long-term treatment of healthy mice with mildronate induces dysbiosis and behavioral deviations despite the effectiveness of mildronate for cardiac and neurological diseases. Thus, we suggest that long-term mildronate treatment is undesirable or at the very least should be accompanied by prebiotics treatments, but this issue should be studied further.